We propose a comprehensive investigation into the relationship between epigenetic variation (DNA Methylation, Histone Methylation and Acetylation), genome-wide, and variation in intravenous drug use in the ALIVE cohort. The AIDS Linked to the Intravenous Experience (ALIVE) study is a prospective community-based cohort of intravenous drug users (IDUs) in Baltimore that commenced in 1988, eventually enrolling more than 3,500 IDUs. Subsequent biennial follow-up included comprehensive assessment of risk behaviors and drug use. We will focus our work on the HIV- subjects in the sample who were already genotyped using the Affymetrix 6.0 platform (N~1200). This sample is >95% African- American addressing a common shortcoming in genomics research. In Aim 1, we propose to examine the relationship between DNA methylation, assessed genome-wide using the Illumina Methylation 450k bead chip, and intravenous drug use, in a subset with available longitudinal biological samples during extended periods of chronic use and then again > 1 year after complete abstinence from all illicit drugs. This is an extension of a preliminary study, presented in the Research Plan, where we found a significant impact of IDU on genome-wide methylation. We will also perform this work on a subsample with subsequent relapse to uncover methylation signals which differ between those who relapse (Aim 1.2) and those who continue to abstain as well as examine the role of methylation in early drug use cessation (Aim 1.3). In Aim 2, we extend this work to examine the impact of long-term chronic use, operationalized as `injection years', on genome-wide methylation signals. Aim 3, we examine the relationship between Histone Methylation (H3K9me2) and Acetylation (H3K9ac) using ChIP Seq technology. As in Aim 1, we will select longitudinal samples from the same individuals during periods of chronic IDU and complete abstinence to maximize the likelihood that we will find differences. We will also attempt to replicate the top findings in a post-mortem brain sample comparing opiate users to controls. This work is novel and innovative for several reasons. The proposed work is among the largest epigenetic studies for any disease or trait in an African American sample. The work builds on a large literature based on animal models of drug exposure for which there are a limited number of human datasets, with available biological samples during periods of chronic use and complete abstinence that could attempt similar work. Lastly, given that we have existing GWAS data, this work will be truly integrative, across many domains of biology (genetic, epigenetic, and phenotypic).